Discordant placental oxygenation and autophagy in twin anemia-polycythemia sequence (TAPS)

Abstract

Background: (Macro)autophagy is an important process of self-degradation of macromolecules and organelles that ensures cellular homeostasis and energy preservation during stressful conditions. Dysregulated placental autophagy has been implicated in a wide range of pregnancy complications. Recent studies identified hypoxia as a key regulator of trophoblast autophagy in vitro; however, its effects on placental autophagy in vivo remain incompletely understood. In this study, we evaluated the monochorionic twin anemia-polycythemia sequence (TAPS) placenta as model of discordant placental oxygenation to determine the effects of hypoxia on placental autophagy in utero.

Methods: We performed a retrospective comparative analysis of tissue oxygenation and autophagy in anemic and polycythemic territories of TAPS placentas (N = 12). Archival tissues were subjected to immunohistochemical, immunofluorescence and Western blot analyses of carbonic anhydrase (CA) IX (hypoxia marker) and key autophagy/lysosomal markers.

Results: CAIX protein levels were significantly higher in anemic twin territories than in corresponding polycythemic territories, consistent with relative tissue hypoxia. Anemic placental shares further displayed significantly higher levels of LC3I/II (autophagosome markers) and LAMP1/2 (lysosome markers), associated with upregulated expression of lysosome/autophagosome activity-associated markers, transcription factor EB and cathepsin D. The accumulation of autophagosomes and lysosomes in anemic shares was accompanied by elevated p62 protein expression, suggestive of inhibition of the downstream autophagy pathway.

Conclusions: TAPS placentas display striking intertwin discordance in tissue oxygenation and autophagic activity and may provide a suitable model for study of the interrelationship between hypoxia, autophagy, and pregnancy outcome in a monochorionic twin setting.

Keywords: Growth restriction; Hypoxia; Monochorionic; Preeclampsia; p62.

Fig. 1.. Representative gross and microscopic appearance of TAPS placenta.

A. Fetal surface following removal of the intertwin membrane and injection of the chorionic vessels. Vascular injection highlights the near-complete separation of the choriovascular beds. A single artery-to-vein anastomosis from anemic (left) to polycythemic (right) territory is shown by arrow. Artery-to-artery and vein-to-vein anastomoses are absent. Color code: Left twin: artery: red, vein: green; right twin: artery: yellow, vein: black. B. Maternal surface demonstrating marked color (redness) discordance with sharp demarcation of anemic (left) and polycythemic (right) territories. C-D. Representative micrographs of plethoric and pale placental parenchyma, respectively (hematoxylin-eosin staining, original magnification ×200).

Fig. 2.. Carbonic anhydrase IX expression in TAPS placentas.

A Western blot analysis of placental carbonic anhydrase (CA) IX expression in lysates of polycythemic (P) and anemic (A) territories of 4 representative TAPS placentas. GAPDH served as loading control. B-C. Representative micrographs of polycythemic and anemic placental parenchyma, respectively (hematoxylin-eosin staining, original magnification ×400) D-E. Representative immunohistochemical analysis of corresponding CAIX protein expression in placental parenchyma, showing intense CAIX immunoreactivity, localized to villous trophoblast, stromal and endothelial cells, in the anemic placental share (right), consistent with relative tissue hypoxia. F-G. Representative immunohistochemical analysis of CAIX protein expression in extravillous trophoblast, showing intense membranous immunoreactivity in the anemic placental share (right). (D-G: DAB-peroxidase system with hematoxylin counterstain, original magnification ×400).

Fig. 3.. Western blot analysis of autophagy-related protein expression in TAPS placentas.

A. Western blot analysis of lysosome- and autophagy-related protein expression in lysates of polycythemic (P) and corresponding anemic (A) territories of 5 representative TAPS placentas. GAPDH served as loading control. B. Densitometric analysis of Western blot. IOD: integrated optical density; *: P < 0.05; **: P < 0.01; ***: P < 0.001 (Wilcoxon matched-pairs signed rank test). LAMP: lysosome-associated membrane protein; LC3: microtubule-associated protein 1A/1B-light chain 3 (LC3-I: cytosolic form; LC3-II: phosphatidylethanolamine conjugate); CSTD: cathepsin D; TFEB: transcription factor EB; A431: human epidermoid carcinoma cell line (ATCC).

Fig. 4.. Immunohistochemical analysis of autophagy-related protein expression in TAPS placentas.

Representative immunohistochemical analysis of expression of LAMP1 (A–B), LAMP2 (C–D), LC3B (E–F) and p62 (G–H) in polycythemic (left) and corresponding anemic (right) territories of TAPS placentas. (DAB-peroxidase system with hematoxylin counterstain, original magnification ×200).

Fig. 5.. Combined immunofluorescence analysis of colocalization of LC3B and LAMP1 in TAPS placentas.

Confocal fluorescence microscopy of polycythemic (A) and anemic (B) territories of TAPS placenta subjected to combined anti-LC3B (red) and anti-LAMP1 (green) immunofluorescence, captured at the same settings. Arrows point to representative regions of colocalization of LC3B and LAMP1, suggestive of fusion of autophagosomes and lysosomes. In selected fields, colocalization was confirmed quantitatively using Pearson’s correlation coefficient analysis whereby a cutoff of r² > 0.5 was used to indicate positive colocalization.